Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

Abstract

The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein

“…Available spike structures with hydrophobic ligands bound in this region indicate that larger pocket openings become accessible after binding. Indeed, cosolvent MD simulations from ligand-free systems showed that aromatic moieties, such as the benzyl group in P2119 and P2165, can bind via induced fit into this intriguing pocket regardless of the glycosylation or conformational state of the RBD (43).…”

Thiol-based mucolytics exhibit antiviral activity against SARS-CoV-2 through allosteric disulfide disruption in the spike glycoprotein

“…Indeed, cosolvent MD simulations from ligand free systems showed that aromatic moieties, such as the benzyl group in P2119 and P2165, can bind via induced fit into this intriguing pocket regardless of the glycosylation or conformational state of the RBD. 43 To explore potential binding modes in greater detail, we evaluated different pocket conformations for docking experiments, including a well-defined pocket due to the presence of a ligand (linoleic acid, PDB 6ZB4) and conformations from our MD simulations (Figure 6a and Table 2, Supporting Information). Using isolated RBD as the receptor template for docking experiments representative of spike open conformations, we found that among the best ranked complex poses were those which allocated P2119 and P2165 to the previously mentioned pocket.…”

Allosteric perspective on the mutability and druggability of the SARS-CoV-2 Spike protein

“…We show that binding to the R3 site causes a large negative allosteric modulation in all RBDs, and results in positive configurational work exerted in the entire S2 subunit. Earlier, it was found that a linear epitope (residue 625-642) in the R3 site may elicit neutralizing antibodies with high specificity (Li et al, 2020b), and a potentially druggable hydrophobic pocket underneath the 617-628 loop, overlapping with the R3 site, was also recently discovered (Zuzic et al, 2021), in addition to the allosteric effects of the order-disorder transition at the 620-640 loop in modulating the RBD updown equilibrium shown in experiments (Zhang et al, 2021). Simulated binding to the R4 site near the junction between the bulk and the stalk of the S2 subunit increases flexibility in the stalk and B.CD while leading to stabilization of the RBD and SD1 of all chains.…”